Many of the physiological activities of a cell are controlled by external signals that stimulate or inhibit intracellular events. The process by which an external signal is transmitted into and within a cell to elicit an intracellular response is referred to as signal transduction. Signal transduction is generally initiated by the interaction of extracellular factors (For example, hormones, adhesion molecules, cytokines, and the like) with membrane receptors on the cell surface. These extracellular signals are transduced to the inner face of the cell membrane, where the cytoplasmic domains of receptor molecules make contact with intracellular targets. The initial receptor-target interactions stimulate a cascade of additional molecular interactions involving multiple intracellular pathways that disseminate the signal throughout the cell. These complex, branching pathways coordinate the multifunctional cellular programs that trigger changes in cell behavior. The orcehstration of diverse proteins in finely tuned intracellular pathways appears to require transient "comparmentalization" of the proteins into complexes. Through a series of inducible and reversible protein-protein interactions, regulatory proteins are recruited from soluble cell material to form short-lived protein complexes that relay signals throughout the cell.
The structural nature of these protein interactions is emerging through the identification of the individual proteins that participate in each signal transduction pathway, the elucidation of the temporal order in which these proteins interact, and the definition or the sites of contact between the proteins. X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy studies have provided detailed structural information on a few of these interactive protein domains.
Many of the proteins involved in signal transduction consist of multiple domains, some of which have enzymatic activity and some of which bind to other cellular proteins, DNA regulatory elements, calcium, nucleotides, or lipid mediators. The discovery that Src homology 2 (SH2) domains provide phosphorylation-dependent and sequence-specific contacts for assembly of receptor signaling complexes has provided a breakthrough in understanding signal transduction (Cantley et al., (1991 ) Cell 64:281-302; Koch et al. (1991 ) Science 252:668-674).
Src homology 2 (SH2) domains were first identified from sequence similarities in the noncatalytic regions of Src-related tyrosine kinases, spanning approximately 100 amino acid residues (Sadowski et al. (1986) Mol. Cell. Biol. 6:4396-4408). The subsequent discovery that SH2 domains bind to specific phosphorylated tyrosine residues has provided a link between tyrosine kinases and proteins that respond to tyrosine phosphorylation (for reviews see Koch et al. (1991) Science 252:668-674; Pawson and Gish, (1992) Cell 71:359-362; Mayer and Baltimore, (1993) Trends Cell Biol. 3:8-13). Now a vast number of proteins likely to be involved in signaling have been shown to contain SH2 domains. The transmission of growth factor-mediated signals, for example, depends critically on the sequence-specific recognition of phosphorylated tyrosines by SH2 domains, which have been discovered in a number of proteins that act downstream of growth factor receptors, including Ras GTPase-activating protein (GAP), phosphatidylinositol 3'-kinase (PIK), and phospholipase C-.gamma. (reviewed by Cantley et al. (1991) Cell 64:281-302). SH2 domains serve to localize these proteins to activated receptors and are implicated in the modulation of enzymatic activity (O'Brien et al. (1990) Mol. Cell. Biol. 10:2855-2862; Roussel et al. (1991) Proc. Natl. Acad. Sci. USA 88:10696-10700; Backer et al. (1992) EMBO J. 11:3469-3479).
While SH2 domains share the common property of binding phosphotyrosine-containing peptides, additional biological specificity resides in the sequence contexts of the phosphotyrosine. Evidence that the binding of a particular SH2 domain to tyrosine-phosphorylated proteins is dependent on the primary sequence around the phosphotyrosine (pTyr) came from a comparison of the sequences of the regions of polyoma middle T and the platelet-derived growth factor (PDGF) receptor that bind phosphatidylinositol 3-kinase (Cantley et al. (1991) Cell 64:281-302). The sequence pTyr-X-Met was found at sites known to be critical for phosphatidylinositol-3-kinase binding to these proteins (Cohen et al. (1990) Proc. Natl. Acad. Sci. 87:4458-4462; Kazlauskas and Cooper, (1989) Cell 58:1121-1133; Talmage et al. (1989) Cell 59:55-65; Whitman et al. (1985) Nature 315:239-242), and this sequence has been predicitve for other receptors or receptor substrates that bind phosphatidylinositol-3-kinase (Lev et al. (1992) Proc. Natl. Acad. Sci. 89:678-682; McGlade et al. (1992) Mol. Cell Biol. 12:991-997; Sun et al. (1991) Nature 352-77; Reedijk et al. (1992) EMBO J. 11:1365-1372). Synthetic phosphopeptides based on this sequence have been found to block phosphatidylinositol 3-kinase binding to the PDGF receptor (Escobedo et al. (1991) Mol. Cell. Biol. 11:1125-1132; Fantl et al. (1992) Nature 352:726-730) and to polyoma middle T (Auger et al. (1992) J. Biol. Chem. 267:5408-5415; and Yoakim et al. (1992) J. Virol. 66:5485-5491). In addition, mutational studies have shown that the SH2 domains of phosphatidylinositol 3-kinase, Ras GAP, and PLC-.gamma. recognize distinct phosphopeptide sequence in the PDGF receptor (Fantl et al. (1992) Cell 69:413-423; Kazlauskas et al. (1990) Science 247:1578-1581; (1992) Mol. Cell Biol. 12:2534-2544).